Type II NADH oxidoreductase (NDH-2), the initial step in the electron transport respiratory chain, is a critical enzyme in the life cycle of Mycobacterium tuberculosis (Mtb), the bacterium that kills more people world-wide than any other bacterial organism. The central role of NDH-2 in Mtb is supported by extensive evidence from biochemical (Weinstein 2005) and transcriptional studies (Boshoff 2005), gene network analysis (Cabusora 2005), gene deletion analysis, investigation of bacterial growth in various media and under various culture conditions (Xie 2005), and animal experiments (Weinstein 2005). Furthermore, isoniazid (INH) and ethambutol (ETH) are two of the standard anti-TB medications used throughout the world and increasing resistance to these medications is recognized as one of the most serious global public health threats. The discovery that a mechanism of INH drug resistance in Mycobacterium tuberculosis is linked to mutations in Mtb NDH-2 that decrease its activity is profoundly important (Miesel 1998;Lee 2001;Vilcheze 2005). Not only is NDH-2 an important enzyme in its biomedical context, it is has certain unique biochemical and structural properties compared to other bacterial type II NADH oxidoreductases. However, very little is known about 1) the catalytic reaction mechanism of wild-type Mtb NDH-2 and INH resistant forms of Mtb NDH-2, 2) the mode of action of inhibitors of the enzyme and 3) the effect of alterations in concentration and activity of NDH-2 on the bioenergetics and biochemistry of the bacterial membrane and the growth phenotypes. Therefore, the overall goals of this grant proposal are (1) to understand the molecular mechanism of the catalytic reaction of wild-type Mtb NDH-2 and INH resistant forms of Mtb NDH-2, (2) to understand the molecular basis of phenothiazine inhibition of these enzymes, and 3) to determine the bacteriologic effects of mutations in NDH-2 and inhibitors of the enzyme in Mtb, M. bovis and M. smegmatis.